Nucleases
Nucleases are enzymes that cleave phosphodiester bonds between the nucleotide subunits of nucleic acids. Deoxyribonucleases act on DNA while ribonucleases act on RNA, however some nucleases utilise both DNA and RNA as substrates.
Nucleases can be further categorised as endonucleases and exonucleases, although some enzymes may have multiple functions and exhibit both endonuclease and exonuclease activity. Endonucleases cleave phosphodiester bonds within a polynucleotide chain. In contrast, exonucleases cleave phosphodiester bonds at the end of a polynucleotide chain. Exonucleases may remove nucleotides from either the 5′ end or the 3′ end or from both ends of a DNA or RNA strand. Flap endonucleases are structure-specific 5′ endonucleases that recognize bifurcated ends of double stranded oligonucleotides and remove single stranded 5′ arms after the first overlapping base leaving a 3′ hydroxyl nick between the two oligonucleotides.
Nucleases are used extensively as tools for molecular biology. Examples of protein endonucleases include restriction endonucleases, Mung Bean nuclease, Endonuclease IV (E. coli), RNase A, RNase I (E. coli), RNase III (E. coli) or RNase H (E. coli). Examples of protein exonucleases include Exonuclease I (E. coli), Exonuclease III (E. coli), Exonuclease VII and T7 Exonuclease. Catalytic nucleic acids including DNAzymes, ribozymes and MNAzymes can also function as endonucleases and cleave phosphodiester bonds within a polynucleotide chain.
Restriction Enzymes
A restriction enzyme (RE) or restriction endonuclease is a catalytic protein that recognizes a specific Restriction Enzyme Recognition (RER) site or sequence (RERS) of a nucleic acid and cleaves the nucleic acid either at the RERS or distant from the RERS. Restriction enzymes are one of the most widely used tools in molecular biology and they are typically purified from bacteria or archaea. For example, EcoRI is purified from Escherichia coli and Hind III is purified from Haemophilus influenzae. Thousands of restriction enzymes have been purified and characterized and greater than 250 different Restriction Enzyme Recognition sequences have been identified.
The type of ends generated by restriction enzyme cleavage include termini where there is a 5′ overhang or a 3′ overhang or the cut may be blunt (no overhang). Most restriction enzymes cleave both strands of a double stranded duplex. Nicking enzymes require a double stranded DNA substrate but only one strand is cleaved. An example of this type of enzyme is Nt.AlwI which recognizes the sequence GGATCNNNN/N and cleaves this strand at the position indicated by the forwardslash (/). Although the majority require a double-stranded DNA as a substrate, a few restriction enzymes have been reported that recognize and cleave single-stranded DNA.
Catalyic Nucleic Acid Enzymes
Catalytic nucleic acid enzymes are enzymes composed of nucleic acid (non-protein enzymes) that can modify nucleic acid substrates. For example, a catalytic nucleic acid enzyme may be a DNA molecule (also known in the art as a DNAzyme or deoxyribozyme or DNA enzyme) or an RNA molecule (known in the art as a ribozyme) or a multi-component nucleic acid enzyme composed of multiple DNA or RNA molecules (known in the art as an MNAzyme). Catalytic nucleic acid endonucleases specifically recognize and cleave distinct nucleic acid substrate sequences. DNAzymes and ribozymes have been shown to be capable of cleaving RNA substrates, DNA substrates and/or chimeric DNA/RNA substrates. Catalytic nucleic acid enzymes can only cleave a nucleic acid substrate (target), provided that the substrate sequence meets minimum sequence requirements. The target substrate must be complementary to the substrate recognition domain (binding arms) of the catalytic nucleic acid and the substrate must contain a specific sequence at the site of cleavage. Examples of such sequence requirements at the cleavage site include the requirement for a purine:pyrmidine sequence for DNAzyme cleavage (10-23 model) and the requirement for the sequence uridine:X where X can equal A, C or U but not G, for the hammerhead ribozymes. The 10-23 DNAzyme is a DNAzyme that is capable of cleaving nucleic acid substrates at specific RNA phosphodiester bonds. This DNAzyme has a catalytic domain of 15 deoxynucleotides flanked by two substrate-recognition domains (binding arms). In the case of DNAzymes and ribozymes, the target substrate sequence that is recognized is the same molecule that is cleaved.
MNAzymes are multi-component nucleic acid enzymes which are assembled and are only catalytically active in the presence of an assembly facilitator. These enzymes are composed of multiple part-enzymes, or partzymes, which self-assemble in the presence of one or more assembly facilitators and form active MNAzymes which catalytically modify substrates. The substrate and assembly facilitators (target) are separate nucleic acid molecules. The partzymes have multiple domains including (i) sensor arms which bind to the assembly facilitator (such as a target nucleic acid); (ii) substrate arms which bind the substrate, and (iii) partial catalytic core sequences which, upon assembly, combine to provide a complete catalytic core. MNAzymes can be designed to recognize a broad range of assembly facilitators including, for example, different target nucleic acid sequences. In response to the presence of the assembly facilitator, MNAzymes modify their substrates. This substrate modification can be linked to signal generation and thus MNAzymes can generate an enzymatically amplified output signal. The assembly facilitator may be a target nucleic acid present in a biological or environmental sample. In such cases, the detection of the modification of the substrate by the MNAyme activity is indicative of the presence of the target. Several MNAzymes capable of cleaving nucleic acid substrates have been reported and additional MNAzymes which can ligate nucleic acid substrates are also known in the art.
Methods Using Restriction Enzymes for Target Detection or Signal Amplification.
Methods using Restriction Enzymes (REs) for detection of target nucleic acid are known in the art. They can distinguish between gene alleles by specifically recognizing single nucleotide polymorphisms (SNPs) in DNA. However, this can only be achieved if the SNP alters the a naturally occurring RERS present in one allele. In this method, the restriction enzyme can be used to genotype a DNA sample without the need for sequencing. Following digestion of genomic DNA with a RE, the resultant DNA fragments can be separated and analysed by gel electrophoresis. In rare instances acquired mutations can be detected if they happen to lie within a naturally occurring RERS.
A number of other methods have been published which exploit RE for target detection using different strategies. One method, known as the Restriction Amplification Assay, uses a labelled oligonucleotide probe which is complementary to the target to be detected and which spans a region of the target that contains a specific RER site (U.S. Pat. No. 5,102,784). Following hybridization of the labelled probe with the target, the resultant duplex is cleaved with a RE and detection of the cleaved probe indicates the presence of the target. Subsequently, another intact probe can bind to a second complementary oligonucleotide and to a cleaved target fragment. This second oligonucleotide binds immediately adjacent to a cleaved target fragment and results in reconstitution of the RE site allowing cleavage of another probe. The disadvantages of this approach include (i) the requirement to have a target containing a specific RERS in the region of interest and (ii) a limited sensitivity, since the maximum number of cleavable duplexes at any time is equal to the original number of target molecules present. The requirement for the target to contain specific RERS in the region of interest significantly limits the flexibility of this assay. The second disadvantage noted above is also of particular importance as the amount of signal-generating complexes present in the assay at any one time is limited to the number of target molecules present which impacts adversely on signal strength and the running time required to achieve satisfactory signal strength. Another example of a target detection assay which employs REs is called the Nicking Endonuclease Signal Amplification (NESA). Similar to the Restriction Amplification Assay, this method employs a labelled oligonucleotide probe which is complementary to the target to be detected and which spans a region of the target that contains a specific RER site, in this case for a nicking RE (Kiesling et al, NAR; 35; 18; e117, 2007). Following hybridization of the labelled probe with the target, one strand of the resultant duplex is cleaved with the nicking RE, and this cleavage results in dissociation of the probe while the target is left intact. Cleavage of the probe generates signal indicative of the presence of the specific target. The target can then hybridize to additional probes causing an increase in the signal. Again, the disadvantages of this approach are (i) the requirement to have a target containing a specific naturally occurring RERS in the region of interest (in this case, specifically the RERS of one of the few nicking RERS adjacent to the target) and (ii) the sensitivity of the approach is limited since the maximum number of cleavable duplexes at any time is equal to the original number of target molecules.
Another protocol, called cascade enzymatic, signal amplification (Zou et al, Angew. Chem. Int Ed; 49 p 1-5; 2010) requires multiple steps, namely; (i) two probes bind to a target creating an overlap that is cleaved by flap endonuclease; (ii) the cleaved flap fragment binds to the loop of a molecular beacon in a position adjacent to a another oligonucleotide also bound to the loop, then T4 ligase joins (ligates) these two oligonucleotides, and this opens the beacon and creates a RERS for a nicking RE; then finally (iii) the nicking RE cleaves the beacon and releases the ligated fragment to bind to another beacon. While this method overcomes the specific need for the nicking RERS to occur naturally in the target, the method teaches that the two fragments must be ligated to create a new RER site. Further, the method is cumbersome, requiring three sequential buffers, one specific for each of the endonclease, ligation and cleavage activity.
None of these methods provide a simple protocol for the amplification of signal generated by the detection of a target in a manner that amplifies the signal independently of the target following an initial target recognition event, regardless of whether or not the specific target has a convenient, naturally occurring RERS.
Other Target and Signal Amplification Technologies
In order to increase the sensitivity of target detection, strategies for target amplification or signal amplification have been employed. Examples of methods which employ target amplification include the polymerase chain reaction (PCR), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), transcript-mediated amplification (TMA); self-sustained sequence replication (3SR), or nucleic acid sequence based amplification (NASBA).
Several examples of signal amplification cascades, which use catalytic nucleic acids, are known in the art. Ligation cascades use a first ribozyme (A) which ligates two RNA containing oligonucleotides to form a second ribozyme (B). Ribozyme (B) then ligates two other RNA containing oligonucleotides to form a new first ribozyme (A), thus triggering a cascade reaction. Other signal amplification cascades use circularized DNAzyme/substrate molecules. A DNAzyme (A) is inactive when circular, but becomes activated by linearization by a second DNAzyme (B), which cleaves the circular DNAzyme (A). Active linear DNAzyme (A) then cleaves circular DNAzyme (B) molecules thus linearizing and activating them. The two DNAzymes capable of cleaving/linearizing each other result in a cascade of catalytic nucleic acid activity.
Other approaches are available including, for example, combining the use of DNAzymes with the versatility of aptamers and/or with the catalytic power of traditional protein enzymes. This method results in the release of a protein enzyme that can, in turn, catalyze the formation of detectable molecules thereby generating and amplifying signal. This approach allows sensitive detection, but it is expensive as it requires highly customized molecules for each assay. Alternate methods include, for example, the branched DNA assay (bDNA) which amplifies a signal by employing a secondary reporter molecule (e.g. alkaline phosphatase) attached to labeled probes mediating the reaction. The Tyramide Signal Amplification (TSA) method uses horseradish peroxidase to convert tyramide to its active form, which binds to tyrosine residues in proteins. The Invader assay allows for nuclease cleavage leading to greater than 1000 cleavage events per target molecule over time. However, there are limitations and deficiencies in known signal amplification methods. For example, the bDNA assay is not as sensitive as the target amplification methods. Apart from sensitivity, known signal amplification assays have been associated with other disadvantages including protracted running time, overly complex protocols and/or increased cost.
Thus, there is an ongoing need for new and improved methods for detecting and quantifying nucleic acid sequences and other targets which incorporate signal amplification.